Gene transfer technique for clinical islet transplantation

研究1。基因转移到大鼠胰岛。从F344大鼠中分离胰岛，培养3-7天后进行基因转移。采用多胺转染试剂进行基因转染。载体为pZeoSV2/lacZ。基因移植胰岛存活5 d，内分泌功能良好。X-gal染色评价基因转移率为90%。研究2。基因转移到犬胰岛。采用1)基因枪、2)电分离、3)脂肪转染的方法将PCMS-EGFP转移至小猎犬胰岛。脂肪抽吸技术操作简单，对胰岛的危害小。此外，脂肪转染技术能很好地保留胰岛素的释放。研究3。临床胰岛分离、冷冻保存和移植。本院共进行了8例胰岛分离，分别使用1例脑死亡供体和7例无心脏跳动供体。胰岛产量为18693 ~ 491040 IEq。纯度为30% ~ 80%。其中一种分离体用于临床同种异体胰岛移植。移植后低血糖消失，血糖水平得到稳定控制。

Study 1.Gene transfer into rat islets. Islets were isolated from F344 rat and cultured for 3-7 days before gene transfer. Gene transfection was made using polyamine transfection reagents. Vector was pZeoSV2/lacZ. Gene transferred islets survived for 5 days with good endocrine function. Gene transfer rate evaluated by X-gal staining showed >90%. Study 2.Gene transfer into canine islets. PCMS-EGFP was transferred into the islats from Beagle pancreata using 1)Gene gun, 2)Electroporarion, and 3)Lipofection. Lipofection technique was simple and less harmful for the isles. Moreover, insulin release was well preserved with Lipofection technique. Study 3.Clinical islet isolation, cryopreservation, and transplantation. Eight human islet isolations were performed in our institute using one brain-dead donor and seven non-heart beating donors. Islet yield was from 18693 to 491040 IEq. Purity was from 30 to 80 %. One of these isolations were used for clinical islet allograft. After transplantation, hypoglycemia disappered and obtained stable control of blood sugar levels.